The concept of using lipid particles as carriers for therapeutic agents has been considered by numerous people. Formulations have relied on complexation of therapeutic agent to the outside of the lipid particle, or actual entrapment of the therapeutic agent, although the ability to make formulations of either type depends on a matching of the characteristics of the lipids and the therapeutic agent, as well as the methods employed to make the particle. In the case of particles with entrapped therapeutic agents, the entrapment method may be passive, i.e., the lipid particles are assembled in the presence of the therapeutic agent, some of which happens to get trapped; or active, i.e, the therapeutic agent is drawn or forced into the interior of a lipid particle as a result of an induced gradient of some type. Notwithstanding the many efforts to utilize lipid particles as carriers, there remain problems which may limit actual applications of lipid-entrapped therapeutic agents. These include low levels of therapeutic agent incorporation on a drug/lipid basis, low efficiency's of capture of the therapeutic agent, and lack of a suitable procedure for larger scale manufacturing of the lipid-encapsulated therapeutic agent particles.
Large scale manufacturing of fully lipid-encapsulated therapeutic agent particles has not been achieved where there is a significant electrostatic interaction between the lipid and the therapeutic agent. A basic problem is aggregation. Aggregation normally results when charged lipid is mixed with oppositely charged therapeutic agent, resulting in a solution containing a milky flocculent mass which is not useable for further processing, let alone for therapeutic use. The aggregation problem has prevented the development of therapeutic compositions which could be of great utility.
Bench scale formulations using charged lipid and oppositely charged therapeutic agent have been successfully achieved using cationic lipids and anionic nucleic acids in a passive encapsulation process described in U.S. Pat. No. 5,705,385 to Bally et al. (PCT Applic. No. WO 96/40964; See also U.S. patent application Ser. Nos. 08/484,282; 08/485,458; 08/660,025; and 09/140,476) and PCT patent Applic. No. WO 98/51278 to Semple et al. (See also U.S. patent application Ser. No. 08/856,374) all assigned to an assignee of the instant invention and incorporated herein by reference. See also Wheeler et al. (1999) Stabilized plasmid-lipid particles: Construction and characterization. Gen. Ther. 6:271–281. These techniques employ an aggregation preventing lipid, such as a PEG-lipid or ATTA-lipid (disclosed in co-pending U.S. patent application Ser. No. 08/996,783 which is incorporated herein by reference), which effectively prevents complex aggregate formation. Resulting fully lipid-encapsulated therapeutic agent particles have excellent pharmaceutical characteristics, such as controlled size (in the 30–250 nm range), full encapsulation (as measured by nuclease resistance, for example) and stability in serum.
WO98/51278 describes a bench scale procedure for the preparation of the lipid-encapsulated therapeutic agent particles using passive entrapment. This known method employs the two basic steps of lipid hydration and liposome sizing. In the lipid hydration step, a cationic lipid solution (95% EtOH solvent) is added dropwise into an agitated reservoir containing polynucleotide therapeutic agent in citrate buffer (pH 3.8) to a final composition of 40% EtOH, 9.9 mg/ml lipid and 2.0 mg/ml polynucleotide. Lipid particles resulting from this hydration step are typically 400 nm diameter and greater, which is too large for general use as a therapeutic. Because of this, extensive post-formulation processing such as high temperature extrusion (at 65° C.) and optionally freeze-thawing (from liquid nitrogen to 65° C. waterbath) is required to obtain suitably-sized lipid particles. The efficiency of encapsulation using this is fairly high (60–90%) in terms of recovered final drug:lipid ratio, but the absolute efficiency of incorporation of starting polynucleotide into the final particle formulation is sub-optimal (25–45%).
Commercial large scale manufacturing of these particles is not efficiently achieved using traditional methods employed in the liposome field. These problems exist notwithstanding the great deal of art on the manufacturing of liposome/drug formulations that has emerged since the first description of liposome preparation by Bangham, AD. et al. (1965) “The action of steroids and streptolysin S on the permeability of phospholipid structures to cations”, J. Mol. Biol. 13, 138–147.
Known large scale manufacturing techniques for lipid particles can be broadly classified into the following categories: 1) Lipid Film Hydration (i.e. Passive entrapment); 2) Reverse Phase Evaporation; 3) High-Pressure extrusion; 4) and Solvent injection (dilution) (see for example U.S. Pat. Nos. 4,752,425 and 4,737,323 to Martin et al). Particular instruments for lipid particle manufacturing disclosed in the art include: U.S. Pat. Nos. 5,270,053 and 5,466,468 to Schneider et al; Isele, U. et al. (1994) Large-Scale Production of Liposomes Containing Monomeric Zinc Phthalocyanine by Controlled Dilution of Organic Solvents. J. Pharma. Sci. vol 83 (11) 1608–1616; Kriftner, R W. (1992) Liposome Production: The Ethanol Injection Technique, in Bruan-Falco et al., eds, Liposome Derivatives, Berlin, Springer-Verlag, 1992, pp. 91–100; Kremer et al. (1977) Vesicles of Variable Diameter Prepared by a Modified Injection Method. Biochemistry 16 (17): 3932–3935; Batzri, S. and Korn, ED. (1973) Single Bilayer Liposomes Prepared Without Sonication, Bioch. Biophys. Acta 298: 1015–1019.
None of the above noted methods or instruments are suitable for scale up of formulations of charged lipid and oppositely charged therapeutic agents with the excellent pharmaceutical characteristics of Bally et al., supra, and Semple et al., supra. The manufacturing techniques set out in Bally et al., supra, and Semple et al., supra were developed only for 1–100 ml preparations, and are cumbersome and lead to unsustainable inefficiencies in large scale manufacturing (i.e. at the scale of 20–200 liters).
The instant invention provides, for the first time, methods for the large-scale preparation of fully encapsulated lipid-therapeutic agent particles where the lipid and therapeutic agent are oppositely charged. These particles are useful as therapeutic compositions and for experimentation and otherwise. It is an object of this invention to provide such methods.